In Wayne's first year, he caught 27 passes for 345 yards with no touchdowns. He only made nine starts and missed three games altogether due to a high ankle sprain. His yards doubled the next year as he caught 49 passes for 716 yards and four touchdowns.

Wayne broke into the starting lineup for good in the 2003 season, and he caught 68 balls for 838 yards and seven touchdowns. The next season, Wayne caught 77 passes for 1,210 yards and 12 touchdowns during a season in which Colts quarterback Peyton Manning set a then-record by throwing for 49 touchdowns. During the playoffs, Wayne was named the "Wild Card Weekend Offensive Player of the Week" after the Colts beat the Broncos. In 2005 Wayne continued to increase his yearly reception total with 83.

In 2006, Wayne signed a 6-year, $39.5 million contract.[10] He went on to catch 86 balls for a then career high 1,310 yards and 9 touchdowns. As a result, Wayne was selected to his first Pro Bowl. Wayne helped the Colts defeat the Chicago Bears in Super Bowl XLI with a 53-yard touchdown reception in the first quarter.

In 2007, Wayne responded to a rash of injuries on the team, including Harrison and tight end Dallas Clark, and set a then career high in receptions (104), and a career high in yards (1510). He led the league in receiving yards and was selected to go to the Pro Bowl for the second consecutive year.

In 2008, Wayne once again posted another Pro Bowl season with 82 catches for 1145 yards and six touchdowns, including a 65-yard score in the opening drive against the Pittsburgh Steelers.

In 2009, Wayne caught the winning touchdown pass against the New England Patriots in what is now known as the "4th and 2" game. Wayne was also selected as a starter for the Pro Bowl, but couldn't play due to the Colts playing in the Super Bowl. In Super Bowl XLIV, Wayne had 5 catches for 46 yards, but the Colts lost to the New Orleans Saints 31-17.

Reggie Wayne cuts vs. the Philadelphia Eagles during the 2010 season.

In 2010, Wayne was second in the NFL receptions with 111 (a career high), and third in receiving yards with 1,355. During a season where the Colts heavily relied on Peyton Manning to win games, Wayne again cemented himself as one of the top receivers in the NFL. Wayne was also selected as a starter the Pro Bowl.

Wayne's numbers were lower than usual in 2011 without Peyton Manning starting at quarterback. Yet still, Wayne led team in receptions (75) and receiving yards (960). He had 4 touchdown receptions as well. On September 25, 2011, Wayne recorded his 800th catch against the Pittsburgh Steelers.

On March 13, 2012, the Colts re-signed Wayne to a three-year contract.[11]

Before the 2012 Week 5 matchup with the Green Bay Packers, the news of head coach Chuck Pagano's leukemia surfaced. Wayne's history with Pagano goes back to his college years, where Pagano was the Hurricanes' defensive backs coach, and Pagano's hiring as the Colts' head coach was a large factor in Wayne's decision to re-sign with Indianapolis. In Week 5, Wayne caught 13 passes for a career high 212 receiving yards and a touchdown from rookie quarterback Andrew Luck. The touchdown came with 35 seconds left and proved to be the winning score. The Colts won 30-27, and Wayne earned the AFC Offensive Player of the Week award due to his performance, the first of his career.

In Week 12 against Buffalo Bills, Reggie Wayne broke Cris Carter's record of consecutive games with 3 or more receptions with 59 games. He also passed former Washington Redskins receiver Art Monk for 12th on the NFL's all-time receptions list during the first half. Monk finished his career with 940 catches.[12] Wayne is currently 8th in receptions among the NFL's all-time reception leaders and 14th on the all-time receiving yards list.

Wayne was selected as the wide receiver for USA Football’s 2012 All-Fundamentals Team, which honors 26 NFL players each year for executing the fundamentals of their position.[14] In a 9–19 loss to the San Diego Chargers on October 14, 2013, Wayne reached 1,000 career receptions, becoming only the ninth receiver to do so. However, a week later, in an October 20 game against the Denver Broncos, Wayne tore his ACL; it was announced the next day that he would miss the remainder of the 2013 season. The injury also ended his consecutive games played streak at 189, the third-longest for a wide receiver in NFL history.[15]

On October 19, 2014, Wayne became the ninth receiver in NFL history to record 14,000 receiving yards.[16] He suffered an elbow injury in the game and missed the following week's matchup against the Pittsburgh Steelers.[17]

On March 6, 2015, the Colts announced that they would not re-sign Wayne, which made him a free agent on March 10.[18]